Diagnosing and monitoring inflammatory diseases by measuring complement components on white blood cells

ABSTRACT

The invention is related to methods of diagnosing inflammatory diseases or conditions by determining levels of components of the complement pathway on the surface of white blood cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 12/511,956,filed Jul. 29, 2009, which is a continuation of U.S. patent applicationSer. No. 10/545,052, filed Oct. 10, 2006, now issued as U.S. Pat. No.7,585,640, which is a U.S. National Stage of International ApplicationNo. PCT/US05/16436, filed May 11, 2005, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 60/570,406, filed May 11, 2004. The priorapplications are incorporated herein in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with United States government support pursuantto Grant Nos. 1 ROI HL074335 and 1 P30 AR47372, from the NationalInstitutes of Health; the United States government has certain rights inthe invention.

FIELD OF THE INVENTION

The invention is related to methods of diagnosing inflammatory diseasesor conditions by determining levels of components of the complementpathway on the surface of white blood cells.

BACKGROUND OF THE INVENTION

This invention relates'to the diagnosis and/or monitoring of patientswith immunologic inflammatory conditions and diseases, e.g., systemiclupus erythematosus (SLE). Inflammation is a characteristic of virtuallyevery immune system disease or condition and every infectious disease orcondition. Many chronic inflammatory conditions and diseases, i.e.,immune system diseases or conditions and infectious diseases orconditions, cause damage to multiple organ systems and are difficult todiagnose. Because the symptoms of many immunologic inflammatoryconditions and diseases overlap there is a great need for diagnosticmethods for rapidly and reliably diagnosing and monitoring specificimmune system disease and conditions. The present invention solves thisand other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for diagnosing or monitoring aninflammatory disease or condition in an individual, determining thelevel of at least one complement pathway component on the surface of awhite blood cell and comparing that determination to the level of thesame complement pathway component on the surface of a white blood cellfrom a control or to a reference value derived from a control whiteblood cell.

In one aspect of the invention, the white blood cells are lymphocytes.Lymphocytes can be isolated using antibodies that recognize specificproteins on the lymphocyte, e.g., using anti-CD3, CD4, CD8 or CD19antibodies.

In another aspect of the invention the inflammatory disease or conditionis e.g., systemic lupus erythematosus (SLE), scleroderma, rheumatoidarthritis, vasculitis, myositis, serum sickness, transplant rejection,sickle cell anemia, multiple sclerosis, gout, pre-eclampsia,cardiovascular disease, and hepatitis C virus infection. The inventionencompasses diagnosis of chronic forms of the above diseases.

In another aspect of the invention, the level of complement componentC4d is determined and used to diagnose or monitor an inflammatorydisease or condition. In some embodiments, levels one or more othercomplements components will be determined in combination with the C4dlevels.

In another aspect of the invention, the level of complement componentC3d is determined and used to diagnose or monitor an inflammatorydisease or condition. In some embodiments, levels one or more othercomplements components will be determined in combination with the C3dlevels.

In one embodiment, the inflammatory disease or condition is SLE. For SLEdiagnosis, the level of complement component C4d is determined on thesurface of lymphocytes. In another embodiment, C4d levels are determinedon the surface of, e.g., a T lymphocyte, a B lymphocyte, or a monocyte.C4d levels can be determined using, e.g., antibodies specific for C4d.The antibodies can labeled for detection and e.g., polyclonal ormonoclonal antibodies can be used.

Diagnosis of SLE can also be accomplished by determining the level ofC4d on a lymphocyte in combination with at least one other complementpathway component. In one embodiment the levels of complement componentsC4d and C3d are determined to diagnose or monitor SLE. C4d levels can bedetermined as above. C3d levels can be determined using, e.g.,antibodies specific for C3d. The antibodies can labeled for detectionand e.g., polyclonal or monoclonal antibodies can be used.

Diagnosis of SLE can also be accomplished by determining the level ofcomplement component C3d on the surface of lymphocytes. In anotherembodiment, C3d levels are determined on the surface of, e.g., a Tlymphocyte, a B lymphocyte, or a monocyte. As above, C3d levels can bedetermined using, e.g., antibodies specific for C3d. The antibodies canlabeled for detection and e.g., polyclonal or monoclonal antibodies canbe used.

In another aspect, the present invention provides a kit for diagnosingor monitoring an inflammatory disease or condition in an individual. Thekit can include an antibody specific for a complement component and ameans for isolating a white blood cell. Generally the means forisolating a white blood cells will be an antibody specific for the whiteblood cell. In one embodiment, the kit includes an antibody that isspecific for complement component C4d. In a further embodiment, the kitincludes a second antibody specific for complement component C3d.

In another embodiment, the white blood cell is a lymphocyte and thelymphocyte is isolated using an antibody specific for the lymphocyte,e.g., anti-CD3, CD4, CD8 or CD19 antibodies. In another embodiment, thewhite blood cell is, e.g., a T lymphocyte, a B lymphocyte, or amonocyte.

In a further aspect the invention provides a computer readable medium,including: (a) code for receiving data corresponding to a determinationof a complement component deposited on surfaces of white blood cells;(b) code for retrieving a reference value for the complement componentdeposited on surfaces of white blood cells of individuals; and (c) codefor comparing the data in (a) with the reference value in (b).

In one embodiment of the computer readable medium, the complementcomponent is C4d. In another embodiment of the computer readable medium,the complement component is C3d. In a further embodiment of the computerreadable medium, the white blood cell is a lymphocyte. In anotherembodiment, the white blood cell is, e.g., a T lymphocyte, a Blymphocyte, or a monocyte.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that complement pathway component C4d is depositedspecifically on peripheral blood T lymphocytes of patients with SLE.Deposition of C4d on peripheral blood T lymphocytes was determined by a2-color flow cytometric assay. T lymphocytes were identified using aFITC-conjugated monoclonal antibody specific for CD3 (a surface markerfor T lymphocytes), and C4d deposited on these cells was determinedusing a monoclonal anti-C4d antibody followed by a PE-conjugatedsecondary antibody. Data shown are C4d-specific mean fluorescence (SMF)of peripheral blood T lymphocytes derived from patients with SLE (n=87,diamonds), patients with other autoimmune diseases (n=31, squares), orhealthy controls (n=19, triangles). A cutpoint of specific meanfluorescence was empirically determined to distinguish individuals withC4d-positive T lymphocytes (SMF>58) from those with C4d-negative Tlymphocytes (SMF<58). The frequencies of individuals with C4d-positive Tlymphocytes among the groups was compared using the Chi-square test, andthe p values for each pair compared are shown above the horizontal line.The mean value of C4d-specific fluorescence on T lymphocytes among thegroups was compared using the Students' T test and the respective pvalues are shown below the horizontal line.

FIG. 2 demonstrates that complement pathway component C3d is depositedspecifically on peripheral blood T lymphocytes of patients with SLE.Deposition of C3d on peripheral blood T lymphocytes was determined by a2-color flow cytometric assay. T lymphocytes were identified using aFITC-conjugated monoclonal antibody specific for CD3 (a surface markerfor T lymphocytes), and C3d deposited on these cells was determinedusing a monoclonal anti-C4d antibody followed by a PE-conjugatedsecondary antibody. Data shown are C3d-specific mean fluorescence ofperipheral blood T lymphocytes derived from patients with SLE (n=87,diamonds), patients with other autoimmune diseases (n=31, squares), orhealthy controls (n=19, triangles). A cutpoint of specific meanfluorescence was empirically determined to distinguish individuals withC3d-positive T lymphocytes (SMFC>3) from those with C3d-negative Tlymphocytes (SMFC<3). The frequencies of individuals with C4d-positive Tlymphocytes among the groups was compared using the Chi-square test, andthe p values for each pair compared are shown above the horizontal line.The mean value of C3d-specific fluorescence on T lymphocytes among thegroups was compared using the Students' T test and the respective pvalues are shown below the horizontal line.

FIG. 3 demonstrates that complement ligands C4d and C3d are depositedspecifically on peripheral blood T lymphocytes of patients with SLE.Deposition of C3d and C4d on peripheral blood T lymphocytes wasdetermined by a 2-color flow cytometric assay. T lymphocytes wereidentified using a FITC-conjugated monoclonal antibody specific for CD3(a surface marker for T lymphocytes), and C3d/C4d deposited on thesecells was determined using a monoclonal anti-C3d (or anti-C4d) antibody,followed by a PE-conjugated secondary antibody. Data shown are C3d- andC4d-specific median fluorescence of peripheral blood T lymphocytesderived from patients with SLE (n=87), patients with other autoimmunediseases (n=31), or healthy controls (n=19). Cutpoints of specific meanfluorescence were empirically determined to distinguish individuals withC3d-positive T lymphocytes (SMFC>3) or individuals with C4d-positive Tlymphocytes (SMFI>58) from those with C3d-negative or C4d-negative Tlymphocytes. The mean value of C3d specific fluorescence on Tlymphocytes among the groups was compared using the Students' T test andthe respective p values are <0.0001 (not shown).

FIG. 4 provides levels of C4d on the Surface of T Lymphocytes, BLymphocytes, and Monocytes. C4d on different types of cells weredetermined by a 3-color flow cytometric assay using monoclonalantibodies specific for cell-specific surface markers and C4d or isotypecontrol immunoglobulins. Levels of C4d were calculated as specificmedian fluorescence intensity (SMFI)=anti-C4d median fluorescenceintensity−isotype Ig median fluorescence intensity. ^(a)T cells wereidentified by electronic gating of cells positively stained by amonoclonal anti-CD3 antibody. ^(b)B cells were identified by electronicgating of cells positively stained by a monoclonal anti-CD19 antibody.^(c)Monocytes were identified by forward and side scattering andnegative staining by anti-Cd3. ^(d)Patients with other inflammatorydiseases such as inflammatory myopathies, Sjogren's syndrome,vasculitis, Raynaud's phenomenon, and cardiovascular disease.^(e)Student t test; patients with SLE vs. patients with other diseases^(f)Student t test; patients with SLE vs. healthy controls.

FIG. 5 provides levels of C3d on the Surface of T Lymphocytes, BLymphocytes, and Monocytes. C3d on different types of cells weredetermined by a 3-color flow cytometric assay using monoclonalantibodies specific for cell-specific surface markers and C3d or isotypecontrol immunoglobulins. Levels of C4d were calculated as specificmedian fluorescence intensity (SMFI)=anti-C4d median fluorescenceintensity—isotype Ig median fluorescence intensity. ^(a)T cells wereidentified by electronic gating of cells positively stained by amonoclonal anti-CD3 antibody. ^(b)B cells were identified by electronicgating of cells positively stained by a monoclonal anti-CD19 antibody.^(c)Monocytes were identified by forward and side scattering andnegative staining by anti-Cd3. ^(d)Patients with other inflammatorydiseases such as inflammatory myopathies, Sjogren's syndrome,vasculitis, Raynaud's phenomenon, and cardiovascular disease.^(e)Student t test; patients with SLE vs. patients with other diseases^(f)Student t test; patients with SLE vs. healthy controls

FIG. 6 Deposition of C4d on Peripheral Blood T cells, depicts the dataof FIG. 1 using a logarithmic scale on the Y-axis providing a clearerpicture of the differences between the healthy controls and the diseasedstates.

FIG. 7 Deposition of C3d on Peripheral Blood T cells, depicts the dataof FIG. 2 using a logarithmic scale on the Y-axis providing a clearerpicture of the differences between the healthy controls and the diseasedstates.

DETAILED DESCRIPTION OF THE INVENTION Introduction

This disclosure provides methods of diagnosing and monitoringinflammatory diseases or conditions by determining the level of at leastone complement component on the surface of a white blood cell.Previously, complement component C4d and CR1 levels on erythrocytes weredetermined and used to diagnose systemic lupus erythematosus (SLE) inindividuals. See, e.g., WO03/022223 published Mar. 20, 2003, which ishereby incorporated by reference for all purposes. This disclosure isthe first to describe diagnosing and monitoring inflammatory diseases orconditions by determining the level of at least one complement pathwaycomponent on the surface of a white blood cell.

In diagnosing the occurrence, or previous occurrence, of an inflammatorydisease or condition, the level of at least one complement pathwaycomponent deposited on surfaces of white blood cells in a sample isdetermined. This determination is then compared with the quantities ofthe same complement pathway component found on the surfaces of whiteblood cells of individuals not having the inflammatory disease orcondition.

In monitoring disease activity of a patient with an inflammatory diseaseor condition, a determination of at least one complement pathwaycomponent is made in the patient's blood sample, and is then comparedwith determinations of the quantities of the same complement pathwaycomponent on surfaces of white blood cells in a sample obtained from thesame patient in the past. Comparison can also be made to quantities ofthe same complement pathway component found on the surfaces of whiteblood cells of individuals not having the inflammatory disease orcondition.

The methods of this disclosure can be used to diagnosis and/or monitorSLE by determining the level of the complement pathway component C4dand/or complement component C3d on lymphocytes. Because SLE is a serioushealth problem, there is a need for relatively accurate and earlydiagnosis of this condition. Likewise, the ability to monitor theactivity of this disease is of great importance. The methods of thisdisclosure can also be used to diagnosis and/or monitor SLE bydetermining the level of C4d and the level of complement pathwaycomponent C3d on lymphocytes.

In diagnosing the occurrence, or previous occurrence, of SLE, complementcomponent C4d alone or in combination with C3d deposited on surfaces oflymphocytes in a sample is determined. This determination is thencompared with the quantities of C4d and C3d found on the surfaces oflymphocytes of individuals not having SLE.

In monitoring disease activity of a patient with SLE, the samedetermination is made in the patient's blood sample, and is thencompared with determinations of the quantities of C4d and C3d onsurfaces of lymphocytes in a sample obtained from the same patient inthe past. Comparison can also be made to quantities of C4d and C3d foundon the surfaces of lymphocytes of individuals not having SLE.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

DEFINITIONS

As used herein, an “inflammatory disease or condition” refers to anyimmune disease or condition that causes increased inflammation in anindividual. An inflammatory disease or condition also refers to anyinfectious disease or condition that causes increased inflammation in anindividual. In some embodiments the inflammatory disease or condition isa “chronic inflammatory disease or condition.” A chronic inflammatorydisease or condition is an inflammatory condition that does not resolveafter a period of weeks, months or longer. Chronic inflammatoryconditions can follow an acute inflammatory condition, or for somediseases or conditions can occur in the absence of an acute inflammatorydisease or condition. An inflammatory disease or condition includes thefollowing: SLE, rheumatoid arthritis, vasculitis (and its specific formssuch is Wegener's granulomatosis), scleroderma, myositis, serumsickness, transplant rejection, sickle cell anemia, gout, complicationsof pregnancy such as pre-eclampsia, multiple sclerosis, cardiovasculardisease, infectious disease such as hepatitis C virus infection, etc.Each of these diseases or conditions can also be described as chronicinflammatory diseases or conditions.

As used herein a “white blood cell” refers to circulating blood cellsthat are not erythrocytes or reticulocytes, e.g., lymphocytes, e.g., Tand B cells, NK cells, eosinophils, basophils, granulocytes,neutrophils, monocytes, macrophages, megakaryocytes, plasma cells,circulating endothelial cells, and stem cells.

As used herein a “control white blood cell” refers to a white blood cellas defined above that is isolated from an individual who does not havean inflammatory disease or condition. When an inflammatory disease orcondition is being monitored in a patient, a control white blood cellcan also refer to a white blood cell isolated from the same patient atan earlier time, e.g., weeks, months, or years earlier.

As used herein the “complement pathway or system” refers to a complexnetwork of more than 30 functionally linked proteins that interact in ahighly regulated manner to provide many of the effector functions ofhumoral immunity and inflammation, thereby serving as the major defensemechanism against bacterial and fungal infections. This system ofproteins acts against invasion by foreign organisms via three distinctpathways: the classical pathway (in the presence of antibody) or thealternative pathway (in the absence of antibody) and the lectin pathway.Once activated, the proteins within each pathway form a cascadeinvolving sequential self-assembly into multimolecular complexes thatperform various functions intended to eradicate the foreign antigensthat initiated the response. For a review of the complement pathway,see, e.g., Sim and Tsiftsoglou, Biochem. Soc. Trans. 32:21-27 (2004).

The classical pathway is usually triggered by an antibody bound to aforeign particle. It consists of several components that are specific tothe classical pathway and designated C1, C4, C2. Sequentially, bindingof C1q to an antigen-antibody complex results in activation of C1r andC1s (both are serine proteases), and activated Cis cleaves C4 and C2into, respectively, C4a and C4b and C2a and C2b. Fragments C4b and C2aassemble to form C4b2a, which cleaves protein C3 into C3a and C3b, whichcompletes activation of the classical pathway. Fragments C4b and C3b aresubject to further degradation by Factor I. This factor cleaves C4b togenerate C4d and also cleaves C3b, to generate iC3b followed by C3d.Thus, activation of the classical pathway of complement can lead todeposition of a number of fragments, such as C4d, iC3b, and C3d, onimmune complexes or other target surfaces. Such targets include cellscirculating in the blood, e.g., lymphocytes and other white blood cells,erythrocytes and platelets.

As used herein a “component of the complement pathway” includes proteinsC1, C4, C2, C3 and fragments thereof, e.g., C1q, C1r, C1s, C4a, C4b,C2a, C2b, C4b2a, C3 a, C3b, C4c, C4d, iC3b, C3d, C3i, C3dg. Alsoincluded are C5, C5b, C6, C7, C8, C9, C1inh, MASP2, CR1, DAF, MCP, CD59,C3aR, C5aR, C1qR, CR2, CR3, and CR4, as well as other complement pathwaycomponents, receptors and ligands not listed specifically herein.

As used herein, “systemic lupus erythematosus”, “SLE”, or “lupus” is theprototypic autoimmune disease resulting in multiorgan involvement. Thisanti-self response is characterized by autoantibodies directed against avariety of nuclear and cytoplasmic cellular components. Theseautoantibodies bind to their respective antigens, forming immunecomplexes which circulate and eventually deposit in tissues. This immunecomplex deposition and consequential activation of the complement systemcauses chronic inflammation and tissue damage.

Diagnosing and monitoring disease activity are problematic in patientswith SLE. Diagnosis is problematic because of the broad spectrum ofdisease ranging form subtle or vague symptoms to life threateningmulti-organ failure. Moreover, other inflammatory diseases withmulti-system involvement can be mistaken for lupus, or vice versa.Criteria were developed for the purpose of disease classification in1971 (Cohen, A S, et al., 1971. Bull. Rheum. Dis. 21:643-648) andrevised in 1982 (Tan, E M, et al., 1982. Arth. Rheum. 25:1271-1277) and1997 (Hochberg, M C. 1997. Arth. Rheum. 40:1725). These criteria aremeant to ensure that patients from different geographic locations arecomparable. Of the eleven criteria, the presence of four or more, eitherserially or simultaneously, is sufficient for classification of apatient as having SLE. Although these criteria serve as useful remindersof those features that distinguish SLE from other related autoimmunediseases, they are unavoidable fallible. Determining the presence orabsence of the criteria often depends on physicians' interpretation andjudgment. The range of clinical manifestations in SLE is much greaterthan that described by the eleven criteria and each manifestation canvary in the level of activity and severity from one patient to another.Furthermore, symptoms of SLE often evolve over the course of disease.There is no definitive test for SLE to date, and, thus, it is oftenmisdiagnosed. This disclosure, however, provides efficient and accuratemethods for diagnosis of SLE and other inflammatory diseases andconditions.

SLE progresses in a series of flares, or periods of acute illness,followed by remissions. The symptoms of a flare, which vary considerablybetween patients and even within the same patient, include malaise,fever, symmetric joint pain, and photosensitivity (development of rashesafter brief sun exposure). Other symptoms of SLE include hair loss,ulcers of mucous membranes and inflammation of the lining of the heartand lungs which leads to chest pain. Red blood cells, platelets andwhite blood cells can be targeted in lupus, resulting in anemia andbleeding problems. More seriously, immune complex deposition and chronicinflammation in the blood vessels can lead to kidney involvement andoccasionally failure requiring dialysis or kidney transplantation. Sincethe blood vessel is a major target of the autoimmune response in SLE,premature strokes and heart disease are not uncommon. Over time,however, these flares can lead to irreversible organ damage.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F (ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F (ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F (ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many techniques known in the art can be used(see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3rd ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No.4,816,567) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized or human antibodies (see,e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992);Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996);Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels for use indiagnostic assays.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to acomponent of the complement pathway or to a marker of a white bloodcell, polymorphic variants, alleles, orthologs, and conservativelymodified variants, or splice variants, or portions thereof, can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with the component of the complement pathwayor the marker of a white blood cell and not with other proteins. Thisselection may be achieved by subtracting out antibodies that cross-reactwith other molecules.

A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988) for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity).

An “antigen” is a molecule that is recognized and bound by an antibody,e.g., peptides, carbohydrates, organic molecules, or more complexmolecules such as glycolipids and glycoproteins. The part of the antigenthat is the target of antibody binding is an antigenic determinant and asmall functional group that corresponds to a single antigenicdeterminant is called a hapten.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, ¹²⁵I, fluorescent dyes, electron-dense reagents,enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, orhaptens and proteins for which antisera or monoclonal antibodies areavailable (e.g., antibody specific for a component of the complementpathway or a marker of a white blood cell can be made detectable, e.g.,by incorporating a radiolabel or fluorescent label into the antibody,and used to detect component of the complement pathway or the marker ofa white blood cell specifically reactive with the labeled antibody). Alabeled secondary antibody can also be used to detect an antibodyspecific for a component of the complement pathway or a marker of awhite blood cell.

The term “contact” or “contacting” is used herein interchangeably withthe following: combined with, added to, mixed with, passed over,incubated with, flowed over, etc.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

In both instances, when speaking of “determination or determining” and“quantity,” we mean to include both an amount or quantity of material.When more than one complement pathway component is measured, e.g., C4dand C3d “determination or determining” and “quantity,” mean in addition,or alternatively, a ratio of a first complement pathway component to asecond complement pathway component, e.g., a ratio of C4d to C3d.

Determination of the Level of a Component of the Complement Pathway on aWhite Blood Cell.

The invention involves conducting assays on white blood cells obtainedfrom patients to determine levels of complement pathway components. Thelevels of the complement components are then used to diagnose or monitoran inflammatory disease or condition in an individual.

Samples of blood are obtained from the patient and are treated with EDTA(ethylenediaminetetraacetate) to inhibit complement activation. Thesamples are maintained at room temperature or under cold conditions.Assays are run preferably within 24 hours.

In some embodiments the white blood cells are isolated from othercomponents of the blood sample. For example, white blood cells (thebuffy coat) can by isolated from plasma and from red blood cells bycentrifugation. Each type of white blood cell can be isolated throughthe use of antibodies against known cell surface markers that arespecific for that cell type, e.g., a lymphocyte. Antibodies against cellsurface markers of white blood cells are known to those of skill and arecommercially available, e.g. from BD Immunocytometry Systems. Forexample, cell surface markers CD3, CD4, CD8, and CD19 are specific forlymphocytes and monoclonal antibodies specific for CD3, CD4, CD8, andCD19 are available from BD Immunocytometry Systems, San Jose, Calif.

Isolation of white blood cells can be done by attaching an antibodyspecific to a cells surface marker to a solid support, then contacting asample containing the white blood cells with the linked antibody.Contaminating cells are washed away allowing the isolated white bloodcells to be collected.

In some embodiments, FACS is used to isolate a white blood cell, e.g., alyphocyte. The term “FACS” refers to fluorescence activated cellsorting, a technique used to separate cells according to their contentof particular molecules of interest. The molecule of interest can bespecific for a type of cell or for particular cell state. The moleculeof interest can be fluorescently labeled directly by binding to afluorescent dye, or by binding to a second molecule, which has beenfluorescently labeled, e.g., an antibody, lectin or aptamer that hasbeen fluorescently labeled and that specifically binds to the moleculeof interest. Thus, white blood cell specific markers can by used toisolate specific white blood cells from other cells in a blood sample.

Isolation of white blood cells also refers to gating techniques used toassay a particular cell population during flow cytometric analysis. Alabeled marker specific for a white blood cell population of interest isused to analyze those cells from a population. A second labeled markeris then used to determine the level of a component of the complementpathway on the surface of the white blood cell.

The determination of the level of a component of the complement pathwaymay be done by a number of methods including flow cytometry, ELISA usingwhite blood cell lysates, and radioimmunoassay. In one embodiment ofthis invention, the determination of the levels of a component of thecomplement pathway is made using flow cytometric methods, withmeasurements taken by direct or indirect immunofluorescence usingpolyclonal or monoclonal antibodies specific for the component of thecomplement pathway. The mean fluorescence channel (MFC) for the whiteblood cell component of the complement pathway is determined.Determination of complement components, e.g., C4d, CR1, and, on thesurface of red blood cells or platelets is described in WO03/022223,published Mar. 20, 2003 and in U.S. Ser. No. 60/463,447, filed Apr. 16,2003, both of which are herein incorporated by reference for allpurposes.

In one embodiment, levels of the complement pathway component C4d orcomplement component C3d are determined on the surface of lymphocytes todiagnose or monitor the progression of SLE in individuals. Thelymphocytes are isolated or detected using lymphocyte specificantibodies e.g., anti-CD3, CD4, CD8, or CD19 antibodies. In someembodiments, complement pathway component C4d and complement pathwaycomponent C3d levels are determined. Determination of C4d and C3d levelscan be done by a number of methods including flow cytometry, ELISA usinglymphocyte lysates, and radioimmunoassay. In one embodiment of thisinvention, the determination of the levels of C4d and C3d is made usingflow cytometric methods, with measurements taken by direct or indirectimmunofluorescence using polyclonal or monoclonal antibodies specificfor C4d or C3d. The mean fluorescence channel (MFC) for lymphocyte C4dor C3d is determined. The same type of assay may be used for diagnosisor for monitoring disease activity in patients known to have SLE.

Kits

Kits for conducting the assays for both the diagnosing of inflammatorydisease and monitoring of inflammatory disease activity are a part ofthis invention. Said kits will use any of the various reagents needed toperform the methods described herein. For example using theimmunofluorescence assays, the kits will generally comprise a conjugateof a monoclonal antibody specific for complement pathway component(e.g., anti-C4d or C3d antibodies) with a fluorescent moiety, andpreferably also a conjugate of a monoclonal antibody specific for awhite blood cell of interest (e.g., lymphocytes using, e.g., anti-CD3,CD4, CD8, and CD19 antibodies) with a different fluorescent moiety.Additionally, the kits can comprise instructional material for the userand such other material as may be needed in carrying out assays of thistype, for example, buffers, radiolabelled antibodies, colorimeterreagents etc.

The antibodies for use in these methods and kits are known. For example,monoclonal antibodies specific for CD3, CD4, CD8, and CD19 are availablefrom Becton Dickinson Immunocytometry Systems, San Jose, Calif. Anti-C4dand anti-C3d antibodies are available from Quidel Corp. in San Diego,Calif. and are generally described in Rogers, J., N. Cooper, et al. PNAS89:10016-10020, (1992); Schwab, C. et al. Brain. Res. 707(2):196 (1996);Gemmell, C. J. Biomed. Mater. Res. 37:474-480, (1997); and, Stoltzner,S. E., et al. Am. J. Path. 156:489-499, (2000).

Diagnostic Methods

Diagnosis of a patient with an inflammatory disease or condition iscarried out by comparing the determination of complement pathwaycomponents with a base value or range of values for the quantities ofthe same complement pathway components typically present on the surfacesof lymphocytes in normal individuals.

For example, diagnosis of a patient with SLE is carried out by comparingthe determination of C4d and/or C3d with a base value or range of valuesfor the quantities of C4d and C3d typically present on the surfaces oflymphocytes in normal individuals. In normal individuals, low levels ofC4d may occasionally be detected but C3d is not present. When using flowcytometric measurement with indirect immunofluorescence, the MFC of C4dand C3d on lymphocytes of healthy individuals ranged from 1.25 to 58.63(mean 17.02) and −3.42 to 2.67 (mean 0.52), respectively. (Tables 1 and3). The MFC of lymphocytes C4d and C3d in patients having SLE was higherthan that of healthy individuals and ranged from −2.62 to 1057.19 (mean201.06) and −3.95 to 318.18 (mean 62.42), respectively (Tables 2 and 3).

Monitoring of Patients

A particular feature of the methods of this invention is to indicate orreflect inflammatory activity that has occurred in the patient duringthe preceding several weeks or even several months. It is possible,using this procedure, to identify the occurrence of a flare-up of aninflammatory disease or condition, such as SLE, during the previous fewweeks or possibly even the previous several months due to persistence ofcomponents of the complement pathway deposited on the surface of whiteblood cells, e.g., C4d and C3d deposited on the surface of lymphocytes.

Automation and Computer Software

The determinations of complement pathway components e.g., C4d and/orC3d, and the diagnostic and disease activity monitoring methodsdescribed above can be carried out manually, but often are convenientlycarried out using an automated system and/or equipment, in which theblood sample is analyzed automatically to make the necessarydetermination or determinations, and the comparison with the base orreference value is carried out automatically, using computer softwareappropriate to that purpose.

Thus, in one aspect, the invention comprises a method for diagnosing ormonitoring an inflammatory disease or condition in an individualcomprising (a) automatically determining, in a blood sample from theindividual containing a white blood cell of interest, complement pathwaycomponents deposited on surfaces of white blood cells in the sample, and(b) automatically comparing said determinations with reference valuesfor the same complement pathway components on surfaces of white bloodcells.

In another aspect, the invention comprises a method for diagnosing ormonitoring SLE in an individual comprising (a) automaticallydetermining, in a blood sample from the individual containinglymphocytes, complement components C4d and C3d deposited on surfaces oflymphocytes in the sample, and (b) automatically comparing saiddeterminations with reference values for components C4d and C3d onsurfaces of lymphocytes.

Computer software, or computer-readable media for use in the methods ofthis invention include:

(1): a computer readable medium, comprising:code for receiving data corresponding to a determination of complementpathway components deposited on surfaces of white blood cells;code for retrieving a reference value for the same complement pathwaycomponents deposited on surfaces of white blood cells of individuals;andcode for comparing the data in (a) with the reference value of (b).

In some embodiments, computer software, or computer-readable media fordiagnosing or monitoring SLE using the methods of this inventioninclude:

(1): a computer readable medium, comprising:code for receiving data corresponding to a determination of complementcomponents C4d and C3d deposited on surfaces of lymphocytes;code for retrieving a reference value for complement components C4d andC3d deposited on surfaces of lymphocytes of individuals; andcode for comparing the data in (a) with the reference value of (b).

In embodiments of the invention, one or more reference values may bestored in a memory associated with a digital computer. After datacorresponding to determinations of complement pathway components isobtained (e.g., from an appropriate analytical instrument), the digitalcomputer may compare the complement pathway component data with one ormore appropriate reference values. After this comparison takes place,the digital computer can automatically determine if the datacorresponding to the determination of t complement pathway component isassociated with an inflammatory disease or condition of interest.

In come embodiments of the invention, one or more reference values maybe stored in a memory associated with a digital computer. After datacorresponding to determinations of complement C4d and C3d is obtained(e.g., from an appropriate analytical instrument), the digital computermay compare the C4d and C3d data with one or more appropriate referencevalues. After this comparison takes place, the digital computer canautomatically determine if the data corresponding to the determinationof complement C4d and C3d is associated with SLE.

Accordingly, some embodiments of the invention may be embodied bycomputer code that is executed by a digital computer. The digitalcomputer may be a micro, mini or large frame computer using any standardor specialized operating system such as a Windows™ based operatingsystem. The code may be stored on any suitable computer readable media.Examples of computer readable media include magnetic, electronic, oroptical disks, tapes, sticks, chips, etc. The code may also be writtenby those of ordinary skill in the art and in any suitable computerprogramming language including, C, C++, etc.

EXAMPLES AND EXPERIMENTAL DATA

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters that could be changed or modified to yieldessentially similar results.

Example 1 Assays of Lymphocyte C4d and C3d in Healthy Controls: Negative

Nineteen healthy individuals were studied. As shown in Table 1, C3d wasnot detected or barely detectable on lymphocytes of each of the nineteenhealthy individuals. Samples of 3 ml of EDTA-anticoagulated peripheralblood were taken from each individual and used as a source oflymphocytes and other white blood cells. The lymphocytes were washed andresuspended in FACS buffer. Levels of C4d, C3d, and CD3 were measured bytwo color indirect immunofluorescence using monoclonal antibodiesspecific for C4d, C3d, and CD3, respectively. Levels of C4d and C3d arequantitated by flow cytometry using a FACSCalibur cytometer (BectonDickinson). The lymphocytes were identified by forward and side scatterand CD3-fluorescence, and the mean fluorescence channel (MFC) wasdetermined for C4d and C3d.

More particularly, blood drawn into a Vacutainer™ containing EDTA(Becton Dickinson, Franklin Lakes, N.J.) was centrifuged at 200×g. Thebuffy coat containing white blood cells (WBC) were carefully collected,transferred into a fresh tube, and washed with phosphate buffered saline(PBS). After removal of contaminating erythrocytes by hypotonic lysis,the remaining leukocytes were washed with PBS and immunofluorescentlyblabeled using different combinations of specific antibodies for atwo-color flow cytometric analysis. Antibodies used in the initial studyincluded: 1) antibodies specific for cell lineage markers, e.g.,anti-CD3, anti-CD4, and anti-CD8 for T lymphocytes, anti-CD19 for Blymphocytes, and anti-CD14 for monocytes (BD PharMingen, San Diego,Calif.), and 2) antibodies reactive to complement C4d or C3d (Quidel,San Diego, Calif.), or the isotype control mouse IgG MOPC21. The stainedcells were then analyzed using a FACSCalibur' flow cytometer and theCellQuest™ software (Becton Dickinson Immunocytometry Systems, San Jose,Calif.). Lymphocytes were electronically gated by forward scatterproperties and expression of CD3 (or CD4, CD8, CD19), specific markersfor lymphocyte subsets. Nonspecific binding of immunoglobulins tolymphocytes was determined by performing identical assays in parallelusing the isotype control antibody MOPC21 (obtained from ATCC). Specificbinding of anti-C4d and anti-C3d were determined by subtracting the MFCobtained with MOPC21 from the MFC obtained with anti-C4d and anti-C3drespectively.

Example 2 Assays of Lymphocyte C4d/C3d to Distinguish Patients with SLEfrom Healthy Controls

This example describes conducting assays on patients to diagnose SLE,and to establish reference values or ranges of values for complementcomponents C4d and C3d.

For this purpose, we recruited 87 patients with SLE from our outpatientoffice. A single determination of lymphocyte C4d/C3d was made in 87individuals who met ACR criteria for the diagnosis of SLE (Table 2) andin 19 healthy controls (Table 1). The mean values of C4d and C3d forpatients with SLE and healthy controls are shown in Table 3. Whereas themean value for C4d and C3d in healthy individuals was 17.02 and 0.52,respectively, the mean value for C4d and C3d among patients with SLE was201.06 and 62.42, respectively (both p=0.0001).

TABLE 1 Levels of C4d and C3d on the Surface of Lymphocytes From HealthyControls Healthy Control C4d Level (SMFC)^(a) C3d Level (SMFC) 200558.23 −2.18 2007 2.36 −1.19 2008 7.9 0.35 2009 1.25 1.81 2022 10.45 1.562003 12.95 2.67 2034 2.95 0.45 2021 20.12 −1.37 2028 5.66 −1.21 20063.34 ND^(b) 2013 7.12 1.62 2011 24.00 −3.42 2037 4.91 −1.97 2010 22.003.11 2025 37.41 1.57 2026 34.55 2.57 2036 22.86 1.55 2029 20.91 1.312035 24.36 2.22 ^(a)specific mean fluorescence channel ^(b)not done

TABLE 2 Levels of C4d and C3d on the Surface of Lymphocytes from SLEPatients SLE Patient C4d Level (SMFC) C3d Level (SMFC) 1039 768.02 68.07107 646.51 253.23 1078 12.71 1.53 1089 0.26 −3.8 1072 12.47 −2.61 101231.48 15.65 1019 30.84 −9.50 1037 0.04 −3.86 1047 355.46 222.75 1003894.69 144.30 1006 ND 133.26 1079 14.80 16.17 1052 548.01 216.67 1038300.77 348.40 1063 20.80 15.09 1095 3.63 33.13 1097 34.73 25.87 109246.44 20.25 1016 352.77 318.28 1093 −2.62 1.06 1094 15.49 6.52 103417.92 8.10 1066 540.76 74.75 1009 1183.30 65.33 1014 141.62 54.80 103182.67 64.40 1086 5.56 −1.29 1098 16.01 0.29 1099 8.79 7.15 1015 1709.54258.91 1100 19.99 18.49 1101 118.94 19.61 1102 191.47 29.13 1053 107.7642.44 1059 108.89 106.60 1084 61.48 29.58 1103 43.90 −1.74 1104 119.494.61 1105 910.93 202.49 1107 44.64 0.00 1109 40.17 17.67 1110 244.7888.44 1111 18.05 5.77 1085 289.48 39.73 1043 6.45 5.46 1056 −1.45 −1.751106 66.56 12.77 1114 241.22 25.51 1017 166.47 43.17 1021 201.31 111.771032 371.08 77.65 1045 4.75 5.23 1115 57.70 20.08 1116 76.77 23.16 111755.45 9.87 1118 48.58 −1.94 1030 50.43 15.72 1061 361.15 165.87 111995.70 29.16 1121 1057.19 263.21 1122 600.58 262.34 1124 23.24 24.69 1125186.13 65.40 1036 763.59 185.30 1044 37.00 12.39 1055 19.95 ND 1126235.13 23.29 1132 745.91 244.91 1133 15.36 −1.62 1136 31.47 12.88 1137314.99 258.50 1138 11.33 0.01 1140 11.98 −0.62 1013 115.8 100.20 1082339.3 49.31 1048 5.07 −2.48 1060 5.96 3.59 1141 5.85 −2.56 1142 17.23−3.95 1143 1.84 0.60 1080 49.98 32.36 1144 −1.76 −0.05 1145 −0.63 −0.671146 82.63 15.68 1147 497.35 227.98 1037 1.71 −1.42 1150 202.73 101.35

Example 3 Assay of Lymphocyte C4d/C3d for Distinguishing Patients withSLE from Patients with Other Diseases

These studies of patients with SLE vs. healthy controls were followed bystudies to compare patients with SLE with patients diagnosed withdiseases other than SLE (n=31; patients with rheumatoid arthritis,scleroderma, or inflammatory myositis). A single determination oflymphocyte C4d/C3d was made, using the same assay (Table 4). The meanvalues of C4d and C3d for patients with SLE, as compared with patientswith other diseases are shown in Table 3. Whereas the mean value for C4dand C3d in patients with other diseases were 29.60 and 12.73,respectively, the mean value for C4d and C3d among patients with SLE was201.06 and 62.42, respectively (both p<0.0001).

TABLE 3 Analysis of Lymphocyte C4d and C3d Levels C4d Level (SMFC)^(a)C3d Level (SMFC)^(b) mean +/− SD range mean +/− SD range SLE 201.06 +/−313.56 −2.62-1057.19 62.42 +/− 89.25 −3.95-318.18 Other Diseases  29.60+/− 55.13     0-263.95 12.73 +/− 29.07 −4.95-141.82 Healthy Control 17.02 +/− 14.86   1.25-58.23  0.52 +/− 1.94 −3.42-2.67 *Leukocytesstained with FITC-anti-CD3 and PE-anti-C4d or PE-anti-C3d were subjectedto 2-color flow cytometric analysis. CD3+ T lymphocytes wereelectronically gated and analyzed for the levels of C4d and C3ddeposited on the surface. ^(a)Specific mean fluorescence intensity(SMFC) of the C4d levels detected on CD3+ T lymphocytes ^(b)SMFC of theC3d levels detected on CD3+ T lymphocytes

TABLE 4 Levels of C4d and C3d on the Surface of Lymphocytes fromPatients with Other Diseases Other Diseases C4d Level (SMFC) C3d Level(SMFC) 17001 2.88 0.24 13010 71.03 10.62 3042 10.11 4.91 4001 8.17 −0.925001 15.83 −0.12 6013 20.37 19.91 6008 10.88 −4.95 6014 19.17 4.38 302212.92 −4.60 4025 26.00 7.2 13032 25.41 56.25 6017 43.70 26.23 4033 11.973.44 4028 173.06 7.98 6011 9.11 2.20 18002 15.33 8.38 4002 85.99 16.518021 4.98 11.22 15005 1.41 −1.71 3029 10.33 0.00 3030 4.41 −3.04 3031263.95 141.82 3032 0.00 −3.12 3034 7.37 70.23 3035 14.01 9.63 4026 0.42−0.24 6002 2.07 −0.52 6008 10.93 2.76 6015 3.98 0.70 4030 21.01 3.1215003 10.88 0.76

Example 4 Assay of Lymphocyte C4d for Distinguishing Patients with SLE

The complement pathway component C4d is deposited specifically onperipheral blood T lymphocytes of patients with SLE. Deposition of C4don peripheral blood T lymphocytes was determined by a 2-color flowcytometric assay. T lymphocytes were identified using a FITC-conjugatedmonoclonal antibody specific for CD3 (a surface marker for Tlymphocytes), and C4d deposited on these cells was determined using amonoclonal anti-C4d antibody followed by a PE-conjugated secondaryantibody. Results are shown in FIG. 1. Data shown are C4d-specific meanfluorescence (SMF) of peripheral blood T lymphocytes derived frompatients with SLE (n=87), patients with other autoimmune diseases(n=31), or healthy controls (n=19). A cutpoint of specific meanfluorescence was empirically determined to distinguish individuals withC4d-positive T lymphocytes (SMF>58) from those with C4d-negative Tlymphocytes (SMF<58). While none of the healthy controls had C4d levelsabove the cutpoint (0/19), almost half of the SLE patients had C4dlevels above the cutpoint (42/87), and some of the patients with otherautoimmune diseases had C4d levels above the cutpoint (4/31). Thefrequencies of individuals with C4d-positive T lymphocytes among thegroups was compared using the Chi-square test, and the p values for eachpair compared are shown above the horizontal line. The mean value ofC4d-specific fluorescence on T lymphocytes among the groups was comparedusing the Students' T test and the respective p values are shown belowthe horizontal line. FIG. 6 presents this data with a logarithmic scaleon the X axis.

Example 5 Assay of Lymphocyte C3d for Distinguishing Patients with SLEand Other Inflammatory Diseases

The complement pathway component C3d is deposited specifically onperipheral blood T lymphocytes of patients with SLE and on patients withother inflammatory diseases. Deposition of C3d on peripheral blood Tlymphocytes was determined by a 2-color flow cytometric assay. Tlymphocytes were identified using a FITC-conjugated monoclonal antibodyspecific for CD3 (a surface marker for T lymphocytes), and C3d depositedon these cells was determined using a monoclonal anti-C4d antibodyfollowed by a PE-conjugated secondary antibody. Results are shown inFIG. 2. Data shown are C3d-specific mean fluorescence of peripheralblood T lymphocytes derived from patients with SLE (n=87), patients withother autoimmune diseases (n=31), or healthy controls (n=19). A cutpointof specific mean fluorescence was empirically determined to distinguishindividuals with C3d-positive T lymphocytes (SMFC>3) from those withC3d-negative T lymphocytes (SMFC<3). While none of the healthy controlshad C3d levels above the cutpoint (0/19), almost 75% of the SLE patientshad C3d levels above the cutpoint (65/87), and about half of thepatients with other autoimmune diseases had C3d levels above thecutpoint (15/31). The frequencies of individuals with C4d-positive Tlymphocytes among the groups was compared using the Chi-square test, andthe p values for each pair compared are shown above the horizontal line.The mean value of C3d-specific fluorescence on T lymphocytes among thegroups was compared using the Students' T test and the respective pvalues are shown below the horizontal line. FIG. 7 presents this datawith a logarithmic scale on the X axis.

Example 6 Summary of Assays Using T-Lymphocytes and Comparison to AssaysUsing B Lymphocytes or Monocytes

Complement ligands C4d and C3d are deposited specifically on peripheralblood T lymphocytes of patients with SLE. FIG. 3 shows deposition of C3dand C4d on peripheral blood T lymphocytes that was determined by a2-color flow cytometric assay. T lymphocytes were identified using aFITC-conjugated monoclonal antibody specific for CD3 (a surface markerfor T lymphocytes), and C3d/C4d deposited on these cells was determinedusing a monoclonal anti-C3d (or anti-C4d) antibody, followed by aPE-conjugated secondary antibody. Data shown are C3d- and C4d-specificmedian fluorescence of peripheral blood T lymphocytes derived frompatients with SLE (n=87), patients with other autoimmune diseases(n=31), or healthy controls (n=19). Cutpoints of specific meanfluorescence were empirically determined to distinguish individuals withC3d-positive T lymphocytes (SMFC>3) or individuals with C4d-positive Tlymphocytes (SMFI>58) from those with C3d-negative or C4d-negative Tlymphocytes. The mean value of C3d specific fluorescence on Tlymphocytes among the groups was compared using the Students' T test andthe respective p values are <0.0001 (not shown). C4d levels weresignificantly higher on T lymphocytes from patients with SLE, ascompared to patients with other diseases or as compared to healthycontrols. C3d levels were also significantly higher on T lymphocytesfrom patients with SLE, as compared to patients with other diseases oras compared to healthy controls.

C4d levels were determined on the surface of T lymphocytes, Blymphocytes, and monocytes of patients with SLE, patients with otherdiseases, and healthy controls. C4d on different types of cells weredetermined by a 3-color flow cytometric assay using monoclonalantibodies specific for cell-specific surface markers and C4d or isotypecontrol immunoglobulins. Levels of C4d were calculated as specificmedian fluorescence intensity (SMFI)=anti-C4d median fluorescenceintensity—isotype Ig median fluorescence intensity. T cells wereidentified by electronic gating of cells positively stained by amonoclonal anti-CD3 antibody. B cells were identified by electronicgating of cells positively stained by a monoclonal anti-CD19 antibody.Monocytes were identified by forward and side scattering and negativestaining by anti-Cd3. Patients with other inflammatory diseases such asinflammatory myopathies, Sjogren's syndrome, vasculitis, Raynaud'sphenomenon, and cardiovascular disease. eStudent t test; patients withSLE vs. patients with other diseases. [C4d levels were significantlyhigher on T lymphocytes, B lymphocytes, and monocytes from patients withSLE, as compared to patients with other diseases or as compared tohealthy controls.

C3d levels were determined on the surface of T lymphocytes, Blymphocytes, and monocytes of patients with SLE, patients with otherdiseases, and healthy controls. C3d on different types of cells weredetermined by a 3-color flow cytometric assay using monoclonalantibodies specific for cell-specific surface markers and C3d or isotypecontrol immunoglobulins. Levels of C4d were calculated as specificmedian fluorescence intensity (SMFI)=anti-C4d median fluorescenceintensity—isotype Ig median fluorescence intensity. T cells wereidentified by electronic gating of cells positively stained by amonoclonal anti-CD3 antibody. B cells were identified by electronicgating of cells positively stained by a monoclonal anti-CD19 antibody.Monocytes were identified by forward and side scattering and negativestaining by anti-Cd3. Patients with other inflammatory diseases such asinflammatory myopathies, Sjogren's syndrome, vasculitis, Raynaud'sphenomenon, and cardiovascular disease. C3d levels were alsosignificantly higher on T lymphocytes, B lymphocytes, and monocytes frompatients with SLE, as compared to patients with other diseases or ascompared to healthy controls.

From the figures and data, it can be seen that complement activationparticipates in a broad range of normal and abnormal inflammatory andimmune processes. Therefore, abnormal patterns of complement activationproducts and complement receptors on white blood cells are useful in thediagnosis and/or monitoring of inflammatory and immune diseases otherthan systemic lupus erythematosus. The data in FIGS. 1-4 support this.Although the highest levels of C3d and C4d on peripheral blood T cellsoccurs in patients with SLE, abnormal levels of C3d are detected on Tcells obtained from 15/31 (48.4%) of patients with other diseases. Inaddition, mean levels of C3d on T cells obtained from patients withother disease are significantly higher than mean levels of C3d on Tcells obtained from healthy controls (p=0.027).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for diagnosing an inflammatory disease in an individual, themethod comprising, (a) quantitating, in a blood sample containing whiteblood cells from the individual, a level of a C4d and/or C3d componentof the complement pathway on a surface of a T lymphocyte, B lymphocyteor monocyte in the sample, and (b) comparing the level in (a) with alevel of C4d and/or C3d with the component of the complement pathway onthe surface of a control T lymphocyte, B lymphocyte or monocyte from anindividual not having the inflammatory disease, wherein an increasedlevel of the C4d and/or C3d component of the complement pathwaydiagnoses the inflammatory disease in the individual.
 2. The method ofclaim 1, wherein the method comprises quantitating, in the blood sample,a level of C4d and/or C3d on the surface of a T lymphocyte, wherein theT lymphocyte is isolated using an anti-CD3 antibody.
 3. The method ofclaim 1, comprising quantitating, in the blood sample, a level of a C4dcomponent of the complement pathway on a surface of a T lymphocyte, Blymphocyte or monocyte in the sample.
 4. The method of claim 1,comprising quantitating, in the blood sample, a level of a C3d componentof the complement pathway on a surface of a T lymphocyte, B lymphocyteor monocyte in the sample.
 5. The method of claim 1, wherein theinflammatory disease or condition is selected from the group consistingof scleroderma, rheumatoid arthritis, vasculitis, myositis, multiplesclerosis, gout, pre-eclampsia, serum sickness, cardiovascular disease,systemic lupus erythematosus (SLE) and hepatitis C virus infection. 6.The method of claim 3, wherein the level of the C4d component of thecomplement pathway is quantitated using an antibody specific for the C4dcomponent of the complement pathway.
 7. The method of claim 6, whereinthe antibody specific for the C4d component of the complement pathway islabeled.
 8. The method of claim 6, wherein the antibody specific for theC4d component of the complement pathway is a monoclonal antibody.
 9. Themethod of claim 3, wherein the level of at least one other complementcomponent is quantitated.
 10. The method of claim 4, wherein the levelof the C3d component of the complement pathway is quantitated using anantibody specific for C3d component of the complement pathway.
 11. Themethod of claim 10, wherein the antibody specific for the C3d componentof the complement pathway is labeled.
 12. The method of claim 10,wherein the antibody specific for the C3d component of the complementpathway is a monoclonal antibody.
 13. The method of claim 9, wherein thecomplement component is C1, C4, C3, C3, C1q, C1r, C1s, C4a, C4b, C2a,C2b, C4b2a, C3a, C3b, C4c, iC3b, C3i, C3dg, C5, C5b, C6, C7, C8, C9,C1inh, MASP2, CR1, DAF, MCP, CD59, C3aR, C1qR, CR2, CR3 or CR4.
 14. Themethod of claim 9, wherein the blood sample is treated withethylenediaminetetraacetate (EDTA) to inhibit complement activation. 15.A method for diagnosing an inflammatory disease in an individual ofinterest, the method comprising, (a) isolating lymphocytes from theindividual of interest using fluorescence activated cell sorting; (b)determining in the lymphocytes, a level of a C4d and/or C3d component ofthe complement pathway on surface of the lymphocytes by determining themean fluorescence channel, and (c) comparing the mean fluorescencechannel in (b) with a mean fluorescence channel of lymphocytes from acontrol individual not having the inflammatory disease, wherein anincreased mean fluorescence channel of the lymphocytes from theindividual of interest as compared to the mean fluorescence channel ofthe control individual diagnoses the inflammatory disease in theindividual of interest.
 16. A computer readable medium, storingcomputer-executable instructions for implementing an evaluation toolusing an automated system, wherein the automated system comprises memoryand a processor, and wherein the evaluation tool evaluates complementcomponent C4d deposits and/or complement component C3d deposits onsurfaces of T lymphocytes, B lymphocytes or monocytes, thecomputer-executable instructions causing the processor to: receive datacorresponding to complement component C4d and/or complement componentC3d deposited on surfaces of T lymphocytes, B lymphocytes or monocytes,store the data corresponding to the complement component C4d and/orcomplement component C3d in the memory of the automated system; storethe retrieved reference value in the memory of the automated system;compare the received data with the reference value; and store results ofthe comparing in the memory of the automated system.
 17. The computerreadable medium of claim 16, wherein the evaluation tool evaluatescomplement component C3d deposits and/or complement component C4ddeposits, and wherein data corresponding to complement component C3d andcomplement component C4d are received.
 18. The computer readable mediumof claim 16, wherein the evaluation tool evaluates C4d deposits and/orC3d deposits on the surface of T lymphocytes.
 19. The computer readablemedium of claim 16, wherein the computer readable medium is read by adigital computer that displays a determination if the complementcomponent C3d deposits and the complement component C4d deposits areassociated with an inflammatory condition
 20. The computer readablemedium of claim 17, wherein the inflammatory condition is systemic lupuserythematosus.